Method and system for standardizing microscope instruments

ABSTRACT

Methods and apparatus for standardizing quantitative measurements from a microscope system. The process includes a calibration procedure whereby an image of a calibration slide is obtained through the optics of the microscope system. The calibration slide produces a standard response, which can be used to determine a machine intrinsic factor for the particular system. The machine intrinsic factor can be stored for later reference. In use, images are acquired of a target sample and of the excitation light source. The excitation light source sample is obtained using a calibration instrument configured to sample intensity. The calibration instrument has an associated correction factor to compensate its performance to a universally standardized calibration instrument. The machine intrinsic factor, sampled intensity, and calibration instrument correction factor are usable to compensate a quantitative measurement of the target sample in order to normalize the results for comparison with other microscope systems.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Divisional of U.S. application Ser. No.12/139,370, filed Jun. 13, 2008, which claims the benefit of U.S.Provisional Patent Application No. 60/944,402, filed Jun. 15, 2007, bothof which are incorporated herein by reference in their entirety.

FIELD

The present invention relates generally to the field of microscopy andmore particularly to standardizing quantitative analytical resultsobtained from the same or different microscope systems to allowcomparisons therebetween.

BACKGROUND

As microscopy platforms become quantitative, a simple method andhardware combination to allow standardization between these platformsneeds to be developed. For example, in fluorescent microscopyapplications, there are products currently available to measurefluorescence intensity for a system (i.e., fluorescent microspheres,fluorescent targets), but they do not provide an overall efficiencyfactor that is related to variations in the overall construction of amicroscope platform, or variations in the light source independent ofsample variations.

SUMMARY

The systems and processes described herein provide normalization factorsfor a given optical microscopy system that can be used to standardizeand scale quantitative measurement results. Standardization allows forcomparison of quantitative results obtained from different instruments,the results from each instrument having undergone the samestandardization. Also, as an extension of this, the same hardware thatcontributes to the instrument efficiency normalization factors can beused to measure variations in light source intensity during anexperiment in which several exposures are taken on a single platformover time.

In one aspect, the invention relates to a process for standardizing aquantitative measurement of target sample data imaged by an opticalsystem. The optical system has an excitation light source, an opticsportion, an image capture portion, and a data storage portion. Thevarious portions of the optical system are cooperatively arranged forobtaining an image of the target sample. A light source correctionfactor is obtained for the excitation light source. The light sourcecorrection factor is applied to the target sample data, therebyobtaining a target sample data standardized with regard to lightintensity variability. A quantitative measure of the standardized targetsample data is determined.

In another aspect, the invention relates to a calibration instrument forsampling illumination of an excitation light source of a microscopysystem. The system includes a calibration surface positioned along anoptical path. The calibration surface substantially uniformly scattersillumination from the excitation light source toward a detection portionof the microscopy system. In some embodiments, the system includes adichromatic mirror positioned to reflect illumination from theexcitation light source along an optical path through an objectivetoward a target sample and to transmit at least a portion ofillumination from the target sample toward a detection portion of themicroscopy system. In such embodiments, the calibration surface ispositioned to temporarily block the optical path between the dichromaticmirror and the objective during calibration and scatter a substantialportion of the reflected excitation light through the dichromatic mirrortoward the detector.

In another aspect, the invention relates to a process for obtaining aquantitative standardized target sample data measurement from an opticalsystem. The optical system has an excitation light source, an opticsportion, a detection portion, and a data storage portion, cooperativelyarranged for obtaining target sample data. An optical system intrinsicfactor is obtained for the optical system. The optical system intrinsicfactor is applied to the target sample data, thereby obtaining a targetsample data measurement standardized with regards to intrinsic opticalfactors.

In another aspect, the invention relates to a process for obtaining astandardized measurement from a microscope system having an excitationlight source, an optics portion, and a detection portion cooperativelyarranged for obtaining an image of a target sample. The process includesilluminating with the excitation light source a calibration sampleconfigured to produce a standard response to the illumination. Acalibration sample image of the illuminated calibration sample obtainedthrough the optics portion is captured with the detection portion. Acalibration instrument configured to direct a sample portion ofillumination from the excitation light source toward the detector isilluminated with excitation light source. An excitation light sourcesample image of the directed sample portion is captured with thedetection portion, and a machine intrinsic factor for correctingvariations along the optical path is determined from the calibrationsample image and the excitation light source sample image. The machineintrinsic factor is usable to compensate a target sample image forintrinsic variations of the microscope system.

In another aspect, the invention relates to a computer-usable mediumhaving computer readable instructions stored thereon for execution by aprocessor performing one or more of the processes described herein.

In another aspect, the invention relates to electromagnetic signalcarrying computer-readable instructions for obtaining a standardizedmeasurement from a microscope system having an excitation light source,an optics portion, and a detection portion cooperatively arranged forobtaining an image of a target sample, in which the instructions performthe process described above.

In another aspect, the invention relates to a microscope systemproviding a standardized measurement, including means for illuminatingwith the excitation light source a calibration sample configured toproduce a standard response to the illumination, means for capturingwith the detection portion a calibration sample image of the illuminatedcalibration sample obtained through the optics portion, means forilluminating with excitation light source a calibration instrumentconfigured to direct a sample portion of illumination from theexcitation light source toward the detector, and means for capturingwith the detection portion an excitation light source sample image ofthe directed sample portion. The system also includes means fordetermining from the calibration sample image and the excitation lightsource sample image a machine intrinsic factor for correcting variationsalong the optical path, the machine intrinsic factor usable tocompensate a target sample image for intrinsic variations of themicroscope system.

In another aspect, the invention relates to a system for compensatingfor intensity measurements of a target sample in a microscope system.The system includes a stage for supporting the target sample, anexcitation light source for illuminating the stage supported targetsample, a detection portion for detecting an image of the illuminatedtarget sample, and a calibration instrument configured for temporaryinsertion along an optical axis between the excitation light source andthe detection portion to redirect a sample portion of the excitationlight source to the detection portion during calibration. Beneficially,the calibration instrument allows for redirection of the excitationlight source without disturbing the staged target sample. The systemalso includes an analyzer in communication with the detection portionfor determining an intensity correction factor determined from theredirected sample portion. The intensity correction factor is usable toadjust detected images of the illuminated target sample to compensatefor excitation light source variations.

In yet another aspect, the invention relates to a process for correctingintensity fluctuations in a fluorescence microscope system having anexcitation light source, an optics portion, and a detection portioncooperatively arranged for obtaining an image of a target sample. Theprocess includes inserting a calibration element in an optical pathbetween an objective and the detection portion. The calibration elementincludes a dichromatic mirror and a calibration surface. The mirror isadapted to reflect light from the excitation light source toward thecalibration surface and to transmit a sample of excitation lightreturned from the calibration surface toward the detector. An intensityvariation of the excitation light source is determined from the sampleof excitation light returned from the calibration surface. Thecalibration element is replaced with a filter set adapted to reflect aselected spectrum of the excitation light source toward the targetsample. A selected spectrum of illumination is transmitted from thetarget sample toward the detector portion. Selected emission lightspectrum is detected from the target sample and the detected emissionlight spectrum from the target sample is corrected using the determinedintensity variation of the excitation light source.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 shows a block diagram of an exemplary microscope system.

FIG. 2 shows a schematic diagram of a calibration instrument constructedin accordance with principles of the present invention.

FIG. 3 shows front, back, left side, right side, top, and bottom viewsof an exemplary calibration instrument constructed in accordance withprinciples of the present invention.

FIG. 4 shows an exploded top perspective view of the calibrationinstrument of FIG. 3.

FIG. 5 shows temporal variation of excitation lamp intensity obtainedfor different calibration instruments in accordance with principles ofthe present invention.

FIG. 6 shows temporal variation of qualitative results for multiplechannels of a first calibration slide.

FIG. 7 shows temporal variation of qualitative results for multiplechannels of a second sample slide.

FIG. 8 shows a flow diagram of a process for standardizing qualitativeanalysis results in accordance with principles of the present invention.

FIG. 9 shows a flow diagram of a process for obtaining correctionfactors used in the standardization of qualitative analysis results ofFIG. 8.

FIG. 10 shows comparative results obtained using different microscopesystems without and with correction accordance with principles of thepresent invention.

FIGS. 11A and 11B show comparison of uncorrected and corrected sampledata obtained in accordance with principles of the present invention.

FIG. 12A shows exemplary qualitative analysis results obtained from thesame sample using different microscope systems without standardization.

FIG. 12B shows correction of the qualitative results of FIG. 12A inaccordance with principles of the present invention.

DETAILED DESCRIPTION

Systems and processes are described herein for obtaining standardizedquantitative analytical results from a system including opticalcomponents and an illumination source. In particular, the systems andprocesses related to standardizing quantitative, microscopic analysis oftarget samples, such as biological samples. Exemplary biological samplesinclude a cell, a group of cells, a tissue section, an array of tissuesections (e.g., a micro tissue array) and combinations of one or more ofany of these. Biological samples can be treated with one or more stains.In some instances, the stains are immunohistochemical stains. In someinstances, the immunohistochemical stains are fluorescent stains.

Generally, a microscope system includes an illumination sourceconfigured to illuminate a target sample, optics configured to produce amagnified image of the illuminated target sample, and a detector, suchas a digital camera, configured to capture a digital image of themagnified image. Quantitative results can be obtained throughmanipulation of the captured digital images. Such image manipulation caninclude image processing techniques known to those skilled in the art.In at least some embodiments, one or more of such image capture andimage manipulation is accomplished with the aid of a processor. Theprocessor can include a computer implementing pre-programmedinstructions.

The system also includes a calibration device configured to redirect astandardized sample of the illumination source to the detector. In atleast some embodiments a system processor is configured to determine acorrection factor for a given microscope. The correction factor can bedetermined from a measurement of the standardized sample of theillumination source obtained using the calibration device. Thecorrection factor can be used (e.g., by the processor) to correct forany variations in intensity of a detected image of the target sample. Insome embodiments, a system processor is configured with instructions(e.g., software) for obtaining the calibration factor. Alternatively orin addition, the system processor is configured with instructions forusing the correction factor to correct detected images. Such calibrationis useful to remove from any quantitative results, variability inintensity of the illumination source within the same microscope system,as may occur over time, and between quantitative results obtained usingdifferent microscope systems and/or different illumination sources.

In some embodiments, the calibration device includes a scatteringsurface positionable along an optical path between the illuminationsource and the detector so as to direct a scattered portion of lightfrom the illumination source toward the detector. Variability in thedetected scattered illumination can be used to develop such a correctionfactor.

In other embodiments a system processor is configured to determine oraccess a correction factor for the optical component of a givenmicroscope. The correction factor can be determined from a measurementof the standardized sample using the optical component of themicroscope. The correction factor can be used (e.g., by the processor)to correct for any variations in optical features of a given microscopeimpacting the intensity of a detected image of the target sample.

Microscope System

Systems and processes described herein are general applicable to anymicroscopy system incorporating an illumination source. Examples of atleast some microscope systems in which the systems and processes can beused included optical microscopy, fluorescent microscopy, and confocallaser scanning microscopy. An exemplary microscopy system is thePM-2000™ instrument commercially available from HistoRx, Inc., of NewHaven, Conn. The systems and processes are particularly useful forsystems geared towards providing a semi-quantitative or quantitativeresult. Exemplary applications include the use of immunohistochemistry(IHC) as used within the field of pathology (See, for example,Immunohistochemistry and Quantitative Analysis of Protein Expression, byM. Cregger et al., Arch. Pathol. Lab. Med., Vol. 130, July 2006 at pgs.1026-1030). Typically, these results are based on the intensity ofstaining of a sample examined using the microscopy system. Samples canbe biological specimens. Stains can be general histological stains,special stains, IHC, FISH, chromogenic, fluorescent, etc.

According to Cregger et al., a diagnostic pathologist typicallyinterprets IHC according to a subjective approach by using a binarypositive-negative end point or a 3- to 4-point scale. With theassistance of a computer, an automated analysis can be obtained fortarget samples using a computer program to help eliminate the inherentvariability of pathologist-based scoring. In immuno-fluorescence, afluorescent product is deposited at the site of an antigen, allowing forvisual localization of the antigen in the sample. After photographiccapture, the reaction product may be quantified by image-analysissoftware. Furthermore the antigen may be located in a specific cellular(e.g., nuclear, organellular, cytoplasmic, membranous) or extra-cellularlocation (See, for example Camp et al, Nature Medicine 8(11) 1323-1327,2002) Numerous computer-based programs have been designed for analysisof IHC, such as BLISS and IHCscore available from Bacus Laboratories,Inc. of Lombard, Ill., ACIS of Clarient, Inc of San Juan Capistrano,Calif., and AQUA® analysis of HistoRx, Inc. of New Haven, Conn.

Generally, for fluorescent IHC, multiple digital images (e.g., TIFF,JPEG, GIF, PNG) are obtained from the same target tissue sample stainedwith protein biomarker-specific antibodies and secondary fluorescentdetection reagents. When optimized, the fluorescent stains provide abroader dynamic range than available by absorbance-based chromogenicstains. Each of the digital images can be obtained using a differentoptical filter configured to pass a respective one of the secondaryfluorescent signals. Thus, at least one respective digital image isobtained for each of the secondary fluorescent signals. For quantitativeanalysis, the captured digital images are manipulated (e.g., using imageprocessing software) to obtain a respective score of the tissue sample.

More specifically, the systems and processes are described in referenceto an exemplary system illustrated in FIG. 1.

Referring to FIG. 1, an exemplary reflected-light fluorescent microscopesystem 100 includes a an excitation source 102, an objective lens 104, asample supporting stage 106, a filter block 108′, and an observationhead 110. The sample supporting stage 106 is configured to support asample under test along an optical axis and within a focal plane of theobjective lens 104. The filter block 108′ is also located along theoptical axis between the objective lens 104 and the observation head110. The filter block 108′ is a three port device with two oppositeports disposed along the optical axis and a third port disposedoff-axis. As illustrated, the third port can be orthogonal to a linejoining the two opposite ports.

Illumination from the excitation source 102 is directed toward theorthogonal port of the filter block 108′. The filter block 108′redirects a portion of the illumination from the excitation source 102toward the objective lens 104. The objective lens 104 preferablyincludes a relatively high numerical aperture thereby allowing it tocapture a substantial portion of excitation light. The objective lens104 functions as a condenser directing excitation light toward a sampleunder test placed upon the stage. In some embodiments, multipleobjective lenses 104 (e.g., 4×, 10×, 20×, 40×, 60×) are included withina single nosepiece (not shown). The nosepiece can be manipulated toselectively bring different ones of the multiple objective lenses 104into alignment with the optical axis to adjust magnification of thesample under test.

Illumination (emission) from the sample under test travels along theoptical path through the objective lens 104 and into a first one of theopposite ports of the filter block 108′. At least a portion of thesample illumination continues along the optical path, exiting a secondone of the opposite ports of the filter block 108′ towards theobservation head 110. As described in more detail below, the filterblock 108′ selectively filters illumination passed therethrough. Influorescence microscopy, filtration can be used to selectively viewemissions from different fluorophores (e.g., red, green, blue). Asillustrated, the microscope system 100 can include multiple filterblocks 108′, 108″, 108′″ (generally 108), each filter block 108 beingtuned to pass a selected emission wavelength toward the observation head110. The different filter blocks 108 can be stored within a carousel orturret 115, allowing for rapid selection of a different filter block 108without disturbing the sample under test. In some embodiments, thedifferent filter blocks 108 are radially disposed within the turret 115about an axis of rotation. The turret 115 is positioned with its axis ofrotation parallel and to a side of the optical axis, such that one ofthe filter blocks 108′ is aligned with the optical axis. Rotation of theturret 115 selectively moves one filter block 108′ out of alignment andbrings another one of the filter blocks 108″, 108′″ into alignment withthe optical axis.

The observation head 110 directs at least a portion of light from thefilter block 108 toward an image collection device, such as a chargecoupled device (CCD) camera 112. In some embodiments, the observationhead 110 additionally includes one or more eyepieces (not shown)allowing for manual observation of the sample under test. Such aneyepiece can be used to adjust placement of a sample 107 upon the stage106 and to coordinate positioning of the stage 106 before and duringtest. In some embodiments, a first shutter 117 is provided to controlexposure time of the sample 107 to the excitation source 102. A secondshutter 114 is provided to control exposure time of an imaging device,such as the CCD camera 112. As shown, the shutter 114 can be anindependent component located along the optical path between the sampleunder test and the observation head 110. Alternatively or in addition toan independent shutter 114, the shutter can be integrated into the CCDcamera 112.

The microscope system 100 also includes a controller 116. The controller116 can be used for controlling the overall image acquisition process.Preferably, the controller 116 is in communication with one or more subelements of the microscope system 110 to allow automated control of thesystem 100. In the exemplary embodiment, the controller 116 is incommunication with one or more of the excitation source 102, an axialtranslator 119 (focus adjust) of the objective lens 104, the CCD camera112, the shutter 114, the turret 115, and a stage positional controller118. The controller 116 can include at least one microprocessor orcomputer 116 operating under the control of program code.

In operation, the controller 116 may send a signal to the stagepositional controller 118 to position the stage 106, such that aselected region or spot 109 of the sample under test is brought intoalignment with the optical axis. The controller 116 may also send asignal to the axial translator 119 configured to position and repositionthe objective lens 104 along the optical axis with respect to the stage106. For embodiments including a motorized nosepiece, the controller 116may send a second signal to the nosepiece causing it to rotate aselected one of multiple objective lenses 104 into alignment with theoptical axis prior to focusing. The controller 116 may also send asignal to the turret 115 causing a controlled rotation of the turret toselect one of the multiple filter blocks 118. In response, the turret118 rotates, bringing the selected one of the filter blocks 118 intoalignment with the optical axis. The controller 116 next sends a signalto the excitation source 102 turning the source 102 on, at leastmomentarily, to illuminate the sample under test. The shutter 114 isnormally closed blocking the optical path between the sample under testand the CCD camera 112. For some microscopes the light source 102 isturned on during initialization of the instrument. With fluorescentmicroscopes, the high-intensity lamps require a warm-up period to allowintensity of the source 102 to stabilize before any test samples aremeasured.

For such fluorescent systems, the light source 102 may remain on duringoperation. In such applications, a first shutter 117 provided betweenlight source 102 and test sample is used to block illumination of thesample until ready to view the sample and acquire an image of thesample. Such limited exposure of the test sample to illumination mayavoid bleaching of the sample. Optionally, a second shutter 114 isprovided within the CCD camera 112. Upon receiving a trigger signal fromthe controller 116, the first shutter 117 opens for a predeterminedexposure period before closing again. A second trigger signal from thecontroller is sent to the second shutter 114 associated with the CCDcamera 112. This signal controls exposure allows a controlled sample ofemission from the sample under test to reach the CCD camera 112. Thus,the first shutter 117 is open for at least the entire duration of anexposure controlled by the second shutter 114. In some embodiments,operation of the two shutters 114, 117 can be controlled by a commonsignal, or otherwise configured to operate in synchronization. Undercontrol of the controller 116, the CCD camera 112 captures an electronicimage of illumination from the sample under test. The image can beforwarded to the controller 116 or to an external system for analysis.

With optional independent control of the two shutters 114, 117, timingof each shutter can be varied to produce different effects. For example,in some embodiments, the first shutter 117 is opened to expose testsample for a predetermined period of time and then closed. This can beperformed to expose a luminescent test sample to illumination from thesource 102. The second shutter 114 could be operated after closure ofthe first shutter 117 to sample luminescence of the sample, withoutinterference from source illumination.

In one particular embodiment, the a fluorescent microscope system ispart of an integrated quantitative IHC analysis system, such as theAQUA® analysis PM-2000™ system, commercially available from HistoRx,Inc. of New Haven, Conn. AQUA is a registered trademark of HistoRx, Inc.The IHC analysis system consists of the following components assembledin a light-tight enclosure: a fluorescent microscope, such as theOlympus BX51 epi-fluorescence microscope, commercially available fromOlympus America, Inc. of Center Valley, Pa.; the microscope is equippedwith a motorized nosepiece to control selection among differentobjective lenses (e.g., 4×, 10×, 20×, 40×, 60×), and a motorized filterturret to control selection among different filter cube selection (e.g.,in DAPI, Cy2, Cy3, Cy5 and Cy7 or equivalent wavelengths). The systemalso includes a motorized stage, such as the Prior Scientific part no.H101A. The PCI card that drives the stage is Prior Scientific part no.H252 motorized stage commercially available from Prior Scientific, Inc.of Rockland, Mass. The control card occupies a PCE expansion slot withina computer controller. Focus control is facilitated by integratedsoftware. The system also includes a light source, such as the X-CITE120 system, commercially available from EXFO Life Sciences & IndustrialDivision of Ontario, Canada, which is equipped with a mercury/metalhalide lamp; a monochromatic digital camera for images capture, such asthe QUANTIFIRE camera, commercially available from OPTRONICS of Goleta,Calif.; and a computer controller. In the exemplary embodiment, thecomputer is a personal computer running WINDOWS XP or higher operatingsystem environment.

Instrument Standardization

In order to standardize quantitative results obtained using a particularsystem, a system intrinsic factor can be determined to account forintensity variability of the excitation source and device variabilityi.e., along the optical path. In order to achieve this, a measurement ofthe intensity of the excitation light source may also be obtained forexample by using an inline lamp intensity measuring tool. Also ameasurement of a standard or a calibration sample i.e., a calibrationmicroscope slide may be obtained using the particular system to defineone or more optical path factors. Use of such a calibration slide isparticularly useful for fluorescence-based IHC applications, in whichsample fluorescent regions of the calibration slide emit radiationwithin respective bandwidths. The fluoresced emissions allow forcharacterization of an optical path at each of the one or morerespective wavelengths. These measurement can be obtained simultaneouslyor separately.

Light Source Intensity Measurement

Generally, a process or instrument to provide for direct measurement ofthe light source intensity is most conveniently incorporated into thesystem. A light source sampling instrument provides for capturing asample of the light source intensity. In some embodiments, a sampledportion of the light source intensity is directed to a detector (e.g., acamera). The light source intensity measurement can be accomplishedindependent of the optical portion of the system.

More generally, the sampling process or instrument accesses a sample ofthe light source at intensity levels below alight source detectorsaturation threshold and above a noise level. For example, the lightsource intensity can be sampled by an electronic sensor within anexposure period (e.g., 10 milliseconds). Alternatively or in addition,the light source can be attenuated to ensure that the obtained samplefalls within the sensitivity range of a given detection device.

The sampled light source intensity can be accomplished using an in lineradiometer, resulting in a measurable voltage representative of thelight source intensity. Such measured voltages can be processedautomatically by the system. For example, the voltages can be sent to aprocessor for further processing. In some embodiments, the voltagelevels are converted into a digital representation of the voltages. Suchconversion can be accomplished using analog-to-digital conversion,allowing for digital processing of the sampled voltage. The digitalprocessing can be accomplished by one or more of software running on theprocessor and hardware adapted for digital signal processing.

The sampled light source intensity can be obtained directly orindirectly from the light source. In some embodiments, the sampled lightsource intensity is obtained independent of at least some other parts ofthe system, such as the optics (e.g., an objective lens), that mayindependently impact the sampled result. Alternatively or in addition,the sampled light source intensity is measured at light source itself,thereby avoiding any effects of the microscope.

Calibration Cube

In some embodiments, a special calibration instrument can be used forthe purpose of obtaining a sample of the light in order to measure theintensity of a light source. Preferably, the calibration instrumentallows a relative light intensity measurement to be obtainedsubstantially simultaneously with the target sample image. In at leastsome embodiments, this is accomplished by switching a specialcalibration instrument into the optical path to obtain the relativelight intensity measurement, and then out of the optical path to obtainthe sample image. For example, if the microscopy system is a fluorescentsystem using multiple filter cubes pre-loaded in a rotatable turret 115,the calibration instrument can be included as one of the filter cubes(i.e., a calibration cube) within the turret 115. This will allow forthe calibration cube to be interchanged with the other filter cubesautomatically during the course of measurements.

Generally a filter cube has openings on the top, bottom, and front facesof a cube-shaped frame. The front opening or port allows light from theillumination source to enter the cube, after which the light isreflected off an internal reflective surface generally positioned at 45degrees to the axis of the entering light. The angled reflective surface(e.g., mirror) redirects a reflected portion of sampled light toward thebottom opening or port of the cube. In operation the redirected lightmay be used to illuminate a target (a tissue sample, etc.). Theredirected light travels along an optical path that may includeobjective optics as provided in microscope systems. At least someportion of the illuminating light may be reflected from the sample. Forat least some applications, stimulated light may also be emitted fromthe sample, as through fluorescence. In either instance, at last aportion of light from the sample (reflected and/or emitted) travels backalong the same optical path, entering the cube from the bottom port. Atleast a portion of the light entering the calibration cube travelsthrough the angled reflective surface of the angled mirror along theoptical path and exits through the top opening or port of the cube andto an imaging device.

In some embodiments, the calibration cube is a modified filter cube inwhich a light scattering surface is affixed to block the bottom opening.In operation, light entering the cube is reflected off the internalangled reflective surface and directed toward the light scatteringsurface. The reflected light illuminates the light scattering surface.At least a portion of scattered light from the light scattering surfaceis directed back up through the internal reflective surface, exiting atthe top opening of the cube toward the imaging device. The samecalibration cube having the same light scattering surface can be used tosample light from the same illumination source at different times and/ordifferent illumination sources. In this manner the calibration cubeprovides a means for sampling light intensity scattered off of astandardized surface (instead of the typical sample) to be captured bythe imaging device, and usable to determine a standardized lightintensity measurement. Beneficially, such sampling can be accomplishedwithout repositioning one or more of the target slide and the objectivelens.

The calibration cube serves as an in-line access tool for measuringintensity of the lamp. In cooperation with a processor 116 (FIG. 1), theintensity measuring tool not only allows for tracking lamp intensitydeviations, but also enables a straightforward means of normalizingquantitative results. Accounting for such lamp intensity deviationspromotes precision measurements of biomarker expression in a tissuesample. For example, data from captured images obtained by severalmicroscopy systems equipped with identically constructed, standardized,calibration cubes, can be corrected to effectively eliminate anycontributions that would otherwise have been attributable to lampintensity variations. Thus, quantitative analysis results, such as AQUAscores obtained from corrected images may be compared for a reliableindication of target sample differences, not system differences. Lightsource calibration data obtained using the calibration cube can becollected, stored and accessed from various system softwareapplications, such as system initialization and setup programs, imageacquisition programs allowing for minimal user interaction andnegligible time and cost.

In one embodiment, referring to FIG. 2, a calibration instrument, orcube 130 includes a housing 132 including a first port 134 a and asecond port 134 b opposite the first and aligned therewith along acommon optical axis. The housing 132 also includes a side port 134 cthat is not aligned with the optical axis. As illustrated, the side port134 c is orthogonal to the optical path. The housing 132 also includesan internal reflective surface 136 forming a nonzero angle θ with theoptical axis. Illumination is received from an excitation source 102through the side port 134 c. The reflective surface 136 is angled toredirect a portion of the received excitation light along the opticalaxis, through the second port 134 b. The calibration cube 130 alsoincludes a light scattering surface 137 positioned relative to thesecond port 134 b to scatter, or return excitation light in an oppositedirection along the same optical axis. At least a portion of thescattered excitation light passes through the reflective surface andexits the housing 132 through the first port 134 a. This scattered lightcan be detected by a CCD camera 112 aligned with the first port 134 a.

In alternative embodiments, a calibration instrument can be formedwithout a mirrored surface. For example, considering the same generalstructure of the cube 130 illustrated in FIG. 2, the internal reflectivesurface 136 can be replaced by a light scattering surface. The lightscattering surface can be angled within the cube to promote redirectionof scattered light from the illumination source 102 through the sideport 134 c.

The light scattering surface 137 is generally uniform, having a surfacethat is a minimally reflective surface (e.g., a matte surface) andprovides uniform reflectance/fluorescence across the field of view whichprovides for also measuring a uniformity of sampled light from the lightsource. In at least some embodiments, the light scattering surface 137scatters light substantially uniformly. Material forming the lightscattering surface 137 should be able to withstand high temperatures andlight intensity without degradation or variation. The material ispreferably rigid or at least semi-rigid and not susceptible toyellowing, degradation, or photo bleaching. Furthermore the materialshould be reproducible, and relatively inexpensive. In some embodiments,certain metallic, ceramic or plastic materials meeting these conditionsare acceptable. Ceramic materials, such as gold and white ceramictargets are commercially available from Avian Technologies, Inc. ofWilmington, Ohio. Such materials can be used for the calibration cubefilter 148 (FIG. 3). Alternatively or in addition, such materials canalso be used for standard calibration slides. In an alternativeembodiment a piece of flat filter paper may be used.

In fluorescent microscope applications, emission light detected by theCCD camera 112 is substantially lower in intensity than the excitationlight. In order to avoid saturation of the CCD camera 112 when detectingthe excitation source itself, one or more filters are included betweenthe excitation source and the camera 112 to attenuate the light to asufficiently low level. In some embodiments, one or more neutraldensity, or gray filters are provided along an optical path between theexcitation source 102 and the CCD camera 112. For example, a firstneutral density filter 138 a is provided at the side port 134 cattenuating excitation light entering the housing 132. A second neutraldensity filter 138 b is provided at the first port 134 a attenuatingscattered light returned to the CCD camera 112. The attenuation valuesof each filter 138 a, 138 b can be the same or different, as long astheir combined effect ensures that the CCD camera 112 will not besaturated by scattered light from the excitation source 102.

EXAMPLE: In an exemplary embodiment of the calibration cube 130 shown inFIG. 3, the cube 130 consists of a regular OLYMPUS filter housing orholder 140 (OLYMPUS Part no. U-M610, U-MF2 BX filter holder cube)equipped with neutral density filters 142 a, 142 b at the emission andexcitation openings 144 a, 144 b and a 50/50 dichroic mirror 146. Insome embodiments, the filters 142 a, 142 b are retained in proximity tothe openings 144 a, 144 b using respective filter frames 145 a, 145 b(FIG. 4).

The bottom of the housing 140 has a light scattering surface 137 (FIG.2); 148 (FIGS. 3, 4) mounted over the port 144 c (FIG. 3) positioned tocompletely block the sample opening 144 c. A top perspective explodedview of the exemplary calibration cube is shown in FIG. 4.

EXAMPLE: In an exemplary embodiment, the calibration cube 130 includestwo neutral density filters, 25 mm, such as Chroma cat. no. 2200a,commercially available from Chroma Technology Corp. of Rockingham, Vt.The cube 130 also includes a dichroic mirror, 50/50 beam splitter, suchas Chroma cat. no. 21000, housed within a filter holder (cube) 140, suchas Chroma cat. no. 91018. The scattering surface can include filterpaper 148, such as VWR cat #28306-153, commercially available from VWRof West Chester, Pa. Which particular brand of filter paper used is notimportant, but preferably the same filter is used among all calibrationcubes 130 of different microscope systems to ensure uniformity ofresults. As will be described below, even this is not critical, asrelative measurements can be made for calibration cubes 130 usingdifferent filter paper 148 compared to a common, or standardizedcalibration cube. Such a comparison can be used to determine an offsetto be accounted for in the correction process.

In other embodiments, the calibration cube 130 includes a microscopefilter holder, such as OLYMPUS Part no. U-M610, U-MF2 BX filter holdercube, commercially available from Chroma Technology Corp of Rockingham,Vt., cat #91018. Other commercially available filter cubes compatiblewith the microscope system may be used. In a particular example, theneutral density filters are ND 1.0 Part no. UVND1.0, ND 1.0 neutraldensity filter, 10% transmittance, 25 mm, and ND 2.0 Part no. UVND2.0,ND 2.0 neutral density filter, 1% transmittance, 25 mm, commerciallyavailable from Chroma Technology Corp. The 50/50 beam splitter/dichroicis Chroma Part no. 21000, 50/50 beam splitter, 38×26 mm. WHATMAN FilterPaper, Grade 1, Cat No. 1001-125, VWR of West Chester, Pa. is affixed tothe bottom of the cube. Beneficially, the filter material scatters anappropriate amount of light back towards the CCD camera 112, such thatan image can be acquired by the camera 112 in a reasonable exposureperiod. For example, the exposure period can be chosen betweenapproximately 3 and 200 milliseconds. Other exposure periods can beselected outside of this range, provided they are appropriate for theapplicable camera capabilities.

Scattered light received at the CCD camera 112 is preferably below thelevel of camera saturation for the exposure time selected withconsideration given to variations in other cube assemblies which may bebrighter or dimmer. Less desirable, but acceptable, is a scatteringmaterial that provides usable signal (below the limit of camerasaturation) for exposure periods greater than about 200 milliseconds.

For use in standardization of systems, the specially designedcalibration filter cube 130 can be installed within the turret of themicroscope system 100 (FIG. 1). This calibration cube 130 serves as aninline lamp intensity measuring tool that provides a means for measuringexcitation light intensity, by sampling scattered light that is directlyproportional to the incident excitation light source intensity. Thus,the sampled scattered light can be used to track variation in lightintensity of the excitation source during use. Such variations inintensity might occur from long-term effects of the excitation sourcesuch as aging, in which intensity of the source may be diminished slowlyduring the normal process of aging. Variations may also result fromshort term effects that may result from other effects, such as ambienttemperature variations, device temperature variations from deviceheating, and excitation voltage and current among others.

During image acquisition in which a sample of the illumination sourcelight intensity is obtained, the light traveling through the excitationneutral density filter 142 a is attenuated, passed through the beamsplitter 146 and reflected off of the calibration material 148 (i.e.,white paper target). Reflected (or scattered) light is then furtherattenuated at the emission neutral density filter 142 b and thencaptured by the camera 112 (FIG. 4). The neutral density filters 142 a,142 b are arranged such that the excitation light is highly attenuatedto reduce the intensity impinging on the calibration material 148. Theemission filter 142 a allows more light through and thus helps reduceintensity observed by the digital camera 112.

Calibration Cube Standardization (CC)

Individual calibration cubes may have intrinsic variations due tomaterial differences that are preferably accounted for in order tonormalize quantitative results obtained across instruments. Inmanufacturing a universal standard cube may be identified. Thereafterall manufactured cubes are compared to the universal standard cube bysampling of a consistent light source with each new cube and anyinherent differences are accounted for, i.e., by applying a cubecorrection or cube calibration factor (CC). The cube calibration factoris preferably determined initially for every new calibration cube. Insome instances, the cube calibration factor can be determinedperiodically thereafter for system maintenance, and when materialproperties of a cube may have changed (i.e., due to aging).

EXAMPLE: In order to characterize a number of similar calibration cubes,results were obtained for each of a batch of five cubes (J1-J5) usingthe same excitation source and camera configuration. A specific one ofthe cubes (i.e., J5) was designated as a reference cube for a group.This cube could be referred to as a universal standard cube. Thereference cube J5, along with the other cubes to be tested, wereinstalled simultaneously into the turret 115 of the microscope system100 (FIG. 1). Images of the sampled illumination from the illuminationsource obtained through the calibration cube 130 were acquired by thedigital camera 112 for each cube J1-J5. Light intensity measurements soobtained were compared between the different calibration cubes beingtested. A ratio of intensity measured for each test cube J1-J4 to theintensity measured using the reference cube J5 was determinedrepresenting a cube calibration factor (CC). In an exemplary experiment,sixteen measurements were collected for each of the different cubesJ1-J5. A ratio of the intensity obtained for each cube J1-J5 tointensity of the reference cube J5 was determined. Table 1 shows the CCvalues determined for the five cubes as compared to the reference cube(J5). Since the construction of the cubes J1-J5 was similar, the ratiosof the intensities are all close to 1.

TABLE 1 Cube Calibration Factor (CC Values) Calibration Cube No. CC CC20.894 J1 0.929 J2 0.989 J3 0.907 J4 0.883 J5 (reference cube) 1.000

To determine temporal effects, light intensity measurements weremeasured for each cube using the same instrument over a two-day period.A minimum of ten measurements were taken through each cube on each day(sum of pixel intensities in the captured image). The results are showngraphically in FIG. 5 as an average total intensity on a scale of 0 to120,000. The results show that light source intensity fluctuations wererecognized in the measurements using all cubes J1-J5. Preferably,correlation coefficient R² values between all cubes are relatively high(e.g., >0.95). The test results suggest that during actual specimenimage acquisition, the intensity of the light source can fluctuate.These fluctuations can occur over a short duration each time the lamp isignited and as slow variations occurring over time while a lamp remainsignited.

Light Source Standardization (LS)

In an exemplary procedure for determining a light source standardizationLS, pixel intensities of a captured image of the illuminated calibrationsurface are combined in a sum. Different ranges can be identifieddepending upon the particular intensity scale values used for thepixels, as well as the number of pixels in the image. Exemplary LSvalues obtained using the AQUA® system range from about of 20,000 to120,000. A ratio can be formed from the LS factor and a chosen intensityvalue. Such a ratio can then be used to compensate target sample data toessentially remove light source variation. In an exemplary embodiment, aratio is formed using a chosen value of 100,000 and an LS factor fallingwithin the AQUA® system range.

Device Optical Path (OP) Measurement

Generally an optical path correction procedure uses a calibration sample(e.g., a calibration slide) providing a known reflectivity and/orfluorescence that is usable in direct measurement of the specific deviceor system's optical path performance. A calibration sample can be usedto obtain a correction factor for the intrinsic optical path performanceof a given microscopy system. When different microscopy systems aresimilarly corrected, target sample results obtained from the differentsystems may be compared reliably across the different microscopysystems.

Most generally the calibration sample has the following characteristics:

-   -   displays some characteristics of the samples typically analyzed        on the system (i.e., for fluorescent systems, these        characteristics can include wavelength of excitement/emission);    -   constructed using a uniform material, with optical properties        (e.g., reflectivity) that are reproducible, available, and        inexpensive;    -   for at least the fluorescent applications, the uniform material        can be opaque (i.e., a ceramic, etc);    -   for bright field transparent applications, the uniform material        attenuates light source, if necessary, to an acceptable level        for the detector; and    -   provides minimal bleaching (for fluorescent systems).

Variations along an optical path of a given microscopy system will notlikely vary to any significant degree over time for the same system.Thus, there is no apparent need to re-perform the optical pathcorrection procedure during normal operations. In at least someembodiments, the optical path correction factor is determined at thetime of manufacture. The optical path correction factor can bere-determined after servicing (e.g., cleaning) of the microscopy system.

Control or Calibration Slide

A standardized instrument calibration sample (control slide orcalibration slide) is used for acquiring data in a particular system tobe calibrated to approximate the light throughput efficiency of aspecific microscope system and optical configuration. For example, thecontrol slide can be a Fluorescent Reference Slide Set XF900, providinga blue or green fluorescent reference slide commercially available fromOmega Optical Inc. of Brattleboro, Vt. Other uniform sample materialsmay be used so long as sufficient signal in each channel (i.e.,wavelength) may be acquired within the set exposure time (e.g., betweenabout 3 and 1000 milliseconds, or current range of the CCD camera 112)and the material preferably demonstrates minimal bleaching over astandard number of runs. The sample material should be reproducible suchthat it can be used for standardizing each instrument and sized to fiton the microscope stage. The material preferably emits or reflects alight signal with spectral components in the appropriate wavelengthband(s) to be acquired through each filter cube in use on the microscopesystem, in an environment of low specular reflection. Other examplesinclude, but are not limited to, alternative colored plastics, paper andceramic reflective materials, metallic surface or surfaces coated withvarious inks and dyes.

EXAMPLE: A calibration slide was placed on the stage of a microscopesystem 100 previously fitted with a pre-standardized calibration cube130 (FIG. 2) in the filter turret 115 (FIG. 1). Two different instrumentcontrol slides were tested: sample 1 having a spectra approximatingFITC/GFP (green excitation) and sample 2 having a spectra approximatingDAPI/Indo/Fura (Blue excitation). Calibration slide 1 was illuminated,and an image obtained of the fluorescence emission of the sample througheach of three different filter cubes 108 (FIG. 1), one for each channeland the calibration cube. Over 900 iterations were performed over aperiod of about thirty hours. Quantitative results, For each channel periteration an intensity score (“derived AQUA® score”) was calculated:mean intensity multiplied by exposure time multiplied by bit depth(i.e., 0-255) The light intensity through the FITC channel when usingsample 1 was too bright indicating this material is not ideal fornormalizing light fluctuations when acquiring in this channel, seeresults described for sample 2 below as an alternative. The results weregraphed as the AQUA analysis score verses iteration for slide 1 (FIG. 6)for calibration slide intensity scores obtained in each Cy3, Cy5channels and separately for the calibration cube. Essentially anidentical light intensity pattern was obtained using instrument controlsample 1 in the Cy3, Cy5 and calibration cube channel indicating thevariability is unlikely due to bleaching of the instrument control slidematerial. Rather, variation is indicative of true light intensityvariability inherent to the system 100. A subtle long-term decrease inquantitative measurements is observable over at least the first half ofthe samples. Superimposed on this are relative short-term variations inboth directions. Interestingly, similar trends are observable in themeasurements obtained for each of the different channels independently,suggesting that the variation is due at least in part to fluctuations inthe intensity of the excitation source.

Light source variability was further tracked by acquiring images of thesecond calibration slide (spectra approximating DAPI/Indo/Fura, blueexcitation) through Cy3, Cy5, and FITC channels, and the calibrationcube over approximately 20 hours for approximately 450 iterations. In anexemplary embodiment, quantitative results, such as the “derived AQUAscores” discussed above, were determined for each iteration. The resultswere graphed as the AQUA analysis score verses iteration for slide 2(FIG. 7). Essentially an identical light intensity pattern resulted fromusing instrument control sample 2 in the Cy3, Cy5 and calibration cubechannel indicating the variability is unlikely due to bleaching of theinstrument control slide material. Rather, variation is indicative oftrue light intensity variability inherent to the system 100. The lightfluctuation pattern seen through the FITC channel when using sample 2was on scale and could be used for standardizing when images are to beacquired in this channel. Modest bleaching of the slide material isevident as a negative slope in the FITC channel over many iterations.Such bleaching is unlikely to adversely impact images acquired undernormal operating conditions. Ideally the instrument control slide isreplaced after about 10% bleaching has occurred. For example after 231runs for sample 1 and approximately 20 runs with sample 2.

Instrument Optical Path Standardization (OP)

The measurement of signal at the digital camera 112 results from lightthat has traveled from the excitation source 102 (FIG. 1) through themicroscope system 100 and has been modified by the intrinsic propertiesof that system 100 and the sample 107 being measured. The optical pathof an instrument 100 may comprise the light pipe which travels from theexcitation lamp to the microscope, the filter cubes 108 and associatedoptics for each fluorescent channel and the objective lens 104 beingused. A machine intrinsic factor (OP) that corrects for variations alongthis optical path can be established for each device 100 in order tostandardize results obtained across devices 100.

To calculate the machine intrinsic factor for a specific system,multiple images (e.g., 16 images) were acquired of a standard instrumentcontrol slide 107. For each system being standardized, the instrumentcontrol slide 107 was of the same material in the sameconfiguration—presumably to yield the same results, but for effects ofthe system 100. Immediately after camera acquisition of a single fieldof view using a specified light filter cube 108 (e.g., FITC, Cy3 or Cy5filter cubes), the filter turret 115 was turned so as to align thecalibration cube 130 (FIG. 2). The calibration cube 130 was separatelyimaged, without disturbing either the objective lens or the sample(i.e., control slide 107) under test. Exposure times for each channelwere fixed. A ratio of the calibration cube intensity to the observedsignal intensity in each channel provided a machine intrinsic factor forthe microscope system optical path efficiency for each filter. Thisvalue is applicable for that system in that specific configuration. Theconfiguration is determined by such features as magnification, lightfilter, and optics.

The machine intrinsic factor of the system can also be scaled byreferencing it to a specific value. The machine intrinsic factor wasdetermined using data obtained using a non-bleached instrument controlslide, at exposure times chosen to avoid saturation, from multiple runs(e.g., five runs) and in independent experiments on different days. Theintrinsic value was calculated for each run. The % CV between runintrinsic values was extremely low such that one run was effective forcalculating intrinsic values.

EXAMPLE: Table 2 shows the resulting machine intrinsic factors for fiveinstruments across filters for three channels: FITC, Cy3 and Cy5.

TABLE 2 Machine Intrinsic Factors (OP Values) Instrument FITC-OP Cy3-OPCy5-OP 1 1.15 1.14 1.09 2 1.61 1.49 1.86 3 1.46 1.38 1.18 4 1.00 1.001.00 5 1.38 1.07 1.32

Machine intrinsic factors were further scaled as the intrinsicvalue/empirical value, where the empirical value was the lowest averagevalue recorded on the particular system. Intrinsic values weredetermined using two different blue instrument control slides and werefound to be reproducible regardless of which slide was used. Averagevalues and percent coefficient of variation (% CV) values werecalculated. Preferably, the % CV is less than about 20%, more preferablythe % CV is less than about 5%.

Standardization

The standardization factors described above (CC, LS, OP) were used totransform quantitative data collected on each individual microscopesystem to that of an idealized system. When applied to more than onesystem, data obtained therefrom are normalized, such that any influenceof the respective microscope and light source fluctuations to theresults were mitigated.

Referring to FIG. 8, a flow diagram of a process for standardizingqualitative analysis results includes a preliminary initializationprocedure 300 followed by a test sample procedure 200. As part of theinitialization procedure 300, the instrument is setup at step 210.Instrument setup includes configuring the microscope system 100 (FIG. 1)with the appropriate excitation source 102, filter cubes 108 (FIG. 1)and calibration cube 130 (FIG. 2), ensuring that the controller 116(FIG. 1) includes the proper program control, and performing anyinitialization routine that may be required for the microscope system100 and CCD camera 112. Once instrument setup is complete, the CCDcamera 112 is capable of obtaining images of a sample under test usingthe microscope system 100 under control of the controller 116. Oncesetup, a correction factor (CC) for the calibration cube 130 and amachine intrinsic factor (OP) for the microscope system 100 are obtainedat step 220. As described above, the machine intrinsic factor (OP) maybe determined at the time of manufacture, and/or at the time ofservicing/repair of the microscopy system and stored for later use.Thus, obtaining the machine intrinsic factor OP may included looking upa pre-stored value. One or more of these factors (CC, OP) can be storedby the controller 116, or image analyzer for later use in analyzingimages of test samples.

As part of the test procedure 200, a sample under test is imaged by thesystem 100 at step 230. A light source correction factor (LS) isobtained during this step. In more detail, an actual test sample 107 isplaced on the microscope stage 106 and positioned such that a targetspot 109 is aligned with an optical axis including the objective lens104 (FIG. 1). This initial alignment can be performed manually throughthe observation head 110 (FIG. 1), automatically using the controller116, or through a combination of a course manual adjustment followed bya fine controller 116 adjustments. For test samples including a regulararray of target spots 109, the test sample 107 is preferably alignedonce (e.g., for one target spot 109) and then re-positioned to testadditional target spots 109 of the sample 107 using preprogrammed offsetadjustments of the stage 106.

Once the target spot 109 is aligned with the optical axis, the system100 acquires a sample image of the target spot 109 using the CCD camera112 (FIG. 1). For the exemplary fluorescence IHC system, the sampleimage is obtained for a chosen wavelength band or channel of interestusing a respective one of the filter blocks 108 (FIG. 1) correspondingto the channel. The calibration cube 130 (FIG. 2) is selected throughrotation of the turret 115 (FIG. 1). A reference image of the excitationsource is also obtained using the calibration cube 130 for adetermination of the excitation source intensity. Additional filtercubes 108 can be used to obtain additional sample images throughdifferent channels, as required. The particular order in which thechannels and excitation source samples are obtained can be varied,provided that the one or more channel images are related to theexcitation source reference image (e.g., taken at approximately the sametime). Such a relationship can be accomplished by forming a compositeimage of the multiple images, or otherwise labeling the images toreflect their relationship.

The sample and reference images can be sent from the camera 112 to thecontroller 116 or separate image analyzer for later analysis andcorrection at step 240. Image analysis can include calculating AQUAscores for each of the different channels. One or more of the correctionfactors (CC, OP, LS) are applied at step 250 to obtain corrected orstandardized results. The standardized output data for the particulartarget spot 109 of the test sample 107 is provided at step 260. The testsample procedure 200 can be repeated for additional target spots 109 ofthe same test sample 107. The test sample procedure 200 can also berepeated for one or more additional test samples 107 using the samecorrection factors obtained at step 220.

In more detail, an exemplary flow diagram of an initialization procedure300 for obtaining correction factors used in the standardization ofqualitative analysis results is shown in FIG. 9. The camera 112 (FIG. 1)and microscope are respectively initialized at steps 310 and 320. Thesesteps may be conducted sequentially or in parallel. Providers of thecamera 112 and microscope system 110 typically define theseinitialization steps 310, 320 in the form of an initializationprocedure. One or more of the initialization procedures may be automatedand occur as part of a power on cycle.

Next, the microscope stage 106 (FIG. 1) is initialized at step 330 toallow for proper alignment of test samples during test. In someembodiments, a calibration slide including a target sample is placedonto the microscope stage 106 at step 350. The calibration slide isselected to provide a known response during characterization of theoptical path, as may be performed at the time of manufacture, orservicing of the microscopy system. An image of the calibration slide isobtained at step 360 and an optical path correction factor, or machineintrinsic factor (OP) is determined at step 370. The machine intrinsicfactor can be stored for later use during normal operation to removeoptical path variability between different microscopy systems. This stepof determining the machined intrinsic factor includes obtaining a sampleimage of the excitation source using the calibration cube 130 (FIG. 2)to determine intensity of the calibration source. Preferably, theexcitation source sample is obtained immediately adjacent to the step ofobtaining an image of the calibration slide to minimize the likelihoodof intensity variation between samples.

Next, the calibration cube correction factor (CC) is accessed at step380. This value can be stored into the system or manually entered duringthe preliminary initialization process 300. This value can be obtainedby comparing results obtained from the calibration cube 130 with resultsobtained using a universally standard calibration cube and formulating aratio of the results. Similar to the optical path correction factor,determination of the calibration cube correction factor need not berepeated during normal use. As described above, the calibration cube ispreferably constructed to reduce or eliminate any variability in itsperformance over time. Thus, an initial determination of the calibrationcube correction factor can be obtained at the time of its manufacture(the factory holds a “gold” standard calibration cube used in comparisonto manufactured calibration cubes to obtain the correction factor). Theresulting correction factor can be marked on the calibration cube itselfand/or provided to the processor for later use during correction ofsampled images. Once obtained, the machine intrinsic factor (OP) andcalibration cube correction factor (CC) are output to image analysissoftware at step 390 (e.g., read from memory locations containingpre-stored values). This can include forwarding the factors (OP, CC) tothe controller 116 or separate image analyzer for storage and later useto standardize images of actual test samples. Steps 360 and 380 can berepeated for different channels of the same calibration slide and outputseparately to the image analysis software at step 390. Thus, the machineintrinsic factor (OP) is determined and retained on a per-channel basisand stored separately for later analysis of test samples on aper-channel basis using the appropriate machine intrinsic factor. Insome embodiments, steps 360, 380, and 390 can be repeated for the samechannel to allow for statistical determination of the machine intrinsicfactor. Thus, multiple results for each channel can be obtained and usedto formulate an average result that is stored for later use.

An equation provided below (EQ. 1) was used to standardize resultsobtained for each system 100. The calibration cube correction factor(CC) and machine intrinsic factors (OP) were determined for each systemand stored for later use in manipulating test results. Next, standardquantitative results (specimen quantitative score_(raw)) were obtainedfor a particular specimen, or sample under test. Through a correctionprocess, the raw quantitative score is multiplied by the variouscorrection factors, to yield a normalized quantitative result suitablefor comparison among different systems.

Specimen Quantitative Score_(normalized)=(specimen quantitativescore_(raw))*(CC)*(OP)*(100,000/LS)  (Eq. 1)

The above equation provided a direct multiplicative standardizationscheme using two constants which were intrinsic to the calibration cubeand microscopy system (CC and OP). Both of these factors were scaled asdescribed above, such that data were standardized to an ideal system.Thus, a calibration cube approximating the “gold standard” would resultin a CC approaching 1.0. Similarly, an optical path approaching astandard reference optical path would also approach 1.0. In the case ofthe light source fluctuation factor (LS), an empirically derived valueof 100,000 was used to define an ideal intensity value for this factor.This value of 100,000 was then divided by the particular LS valueobtained during each measurement. Table 3 shows the resultingstandardization correction factor obtained for five differentinstruments.

TABLE 3 Standardization Factor for Five Instruments InstrumentCorrection: (CC) * (OP) * (100,000) 1 97446 2 116702 3 172794 4 88300 5132000

Using the correction factors described herein (CC, OP, LS), thequantitative data obtained using the five different instruments were allcorrelated to instrument 1. The correlation results are illustratedgraphically in FIG. 10. As can be seen from the figure, beforecorrection, the correlations of each instrument to instrument 1 resultin non-overlapping curves varying substantially from instrument 1. Afterapplication of the correction factors, however, the correctedcorrelation curves for all five instruments to instrument 1 overlapsubstantially, demonstrating a high degree of correlation to instrument1 and to each other.

EXAMPLE: In use, a test sample can include a tissue microarray (TMA)including a matrix of tissue samples on a single microscope slide. Forexample, a 36 spot tissue microarray including breast cancer tissuesamples, BT474, MCF7, T47D cell line control samples was stained. Thestaining protocol involved deparafinization in xylene, rehydrationthrough a series of decreasing amounts of ethanol to pure water, andantigen retrieval in Tris EDTA. After endogenous peroxidase blocking andblocking with background sniper, HER 2, (CB11) and cytokeratin (Rabbit,Dako) primary antibodies were applied and rinsed off after 1 hour. DakoEnvision anti-mouse and Invitrogen alexa 555 GAR were then applied.After extensive washing, cy 5 tyramide was applied. The slides were thenwashed in TBS/Tween 20. Finally, a mounting media with Dapi was appliedand the slides were dried.

The fluorescent intensity of staining for HER2 and resulting AQUA scorewere collected for each tissue spot of the tissue micro array usinginstruments 1 and 2 included in Table 3.

Scores were standardized using Eq. 2 and Eq. 3 below.

Quantitative Score_(normalized)=(QuantitativeScore_(raw Instrument 1)/LS_(Instrument 1))*97,446.  (Eq. 2)

Quantitative Score_(normalized)=(QuantitativeScore_(raw Instrument 2)/LS_(Instrument 2))*116,702.  (Eq. 3)

AQUA scores for each sample in the 36 spot tissue micro array wereacquired using two different instruments. The raw AQUA scores shown inFIG. 11A and the normalized AQUA scores shown in FIG. 11B from the 36spot tissue micro array, acquired on two instruments were graphed inscatter plot format. The results show a slope of the regression line ofthe correlation coefficient of 2.6:1 between the two instruments usingthe raw scores. Using the standardization methods of the invention, aslope of the regression line of the correlation coefficients of 0.98:1between the two instruments was achieved showing that the distributionof scores are not effected by standardization.

Non-parametric Spearman's rho statistical analysis was used to describethe ranking relationship before and after standardization. The analysiswas performed using the AQUA score data set obtained from twoinstruments. Rank-orders are assigned from smallest original value(=rank 1) to highest original value for each AQUA score. The correlationcoefficient and the P-Value were calculated for the standardized and thenon-standardized (raw) datasets. The rank order of the data wasunaffected by standardization (Table 4).

TABLE 4 Spearman Rho analysis Normalized Data Raw Data Rho: 0.72 0.721P-Value: <0.0001 <0.0001

EXAMPLE: The same 36 spot stained tissue micro array described in theabove example was acquired and scored on five different instruments.

The results indicated that the percent coefficient of variation (% CV)of the raw AQUA scores and the standardized AQUA scores for each tissuemicro array tissue spot acquired on the five different instruments showssignificantly better % CV is achieved by standardization by the methodsof the invention.

Tables 5 and 6 are compilations of the slope of the regression line ofthe correlation coefficient generated with a single slide run on thefive instruments. Correlations based on raw AQUA scores are shown inTable 5 and those based on standardized AQUA scores are shown in Table6. This comparison was done with numbers generated from validatedimages.

TABLE 5 Raw data Instrument Instrument Instrument Instrument InstrumentRaw Data 1 2 3 4 5 Instrument 1 N/A 0.44x 2.55x 1.68x 2.51x Instrument 20.44x N/A 0.17x 3.85x 5.76x Instrument 3 2.55x 0.17x N/A 0.66c 0.98xInstrument 4 1.68x 3.85x 0.66x N/A 1.50x Instrument 5 2.51x 5.76x 0.98x1.50x N/A

TABLE 6 Standardized data Normalized Instrument Instrument InstrumentInstrument Instrument Data 1 2 3 4 5 Instrument 1 N/A 0.88x 0.95x 1.09x0.89x Instrument 2 0.88x N/A 0.92x 1.24x 1.00x Instrument 3 0.95x 0.92xN/A 1.14x 0.93x Instrument 4 1.09x 1.24x 1.14x N/A 1.23x Instrument 50.89x 1.00x 0.93x 1.23x N/A

The percent coefficient of variation (% CV) of the raw AQUA® scores andthe standardized AQUA scores for each tissue micro array tissue spotacquired on five instruments shows significantly better % CV is achievedby standardization by the methods of the invention.

The mean AQUA scores for HER2 staining of 26 validated tissue microarray spots of the 36 are shown in FIG. 12A and FIG. 12B. While trendsor comparisons across the 36 samples are similar on each instrument,score variance for each individual sample have an average CV ofapproximately 60% from instrument to instrument. In comparison the meanscores obtained on each of the same 5 instruments, once standardizedFIG. 12B are more consistent, with an average CV of approximately 20%.

An analysis of variance (ANOVA) test for significant differences betweenmeans before and after standardization using the null hypothesis thatthe instruments are identical produces a significant p value <0.05 thusrejecting the null hypothesis indicating the marginal mean scorescollected on multiple instruments are different. After standardizationthe p value of >0.05 indicates that marginal mean scores collected onmultiple instruments is now not significantly different.

Although the exemplary embodiments relate primarily to fluorescentmicrocopy, the invention is broadly applicable to optical microscopes ingeneral. The techniques of various embodiments apply to correcting forvariations in intensity of any light source using a calibrationinstrument, correcting for calibration instrument variations usingoffsets to a universally standard calibration instrument, and/ornormalizing effects of the optical path through a particular opticalmicroscope system. Thus, various embodiments of the invention can beapplied generally with a variety of optical microscopes using incoherentillumination sources, polarized illumination sources, and coherentillumination sources, such as confocal laser scanning microscopesystems.

System requirements for an exemplary fluorescent microscopy system areincluded in Table 7.

TABLE 7 Exemplary Fluorescent Microscopy System Requirements ComponentTarget Specifications Example Microscope Epi-fluorescence microscopeOlympus BX51 Stage automation to facilitate image HistoRx PM2000 ™system acquisition (optional) (Prior Stage) with associated AQUAsition ™software Mercury (Hg) arc fluorescence light Exfo X-cite with adjustableiris source. It is strongly recommended that light sources possess anadjustable iris or safety shutter if light measurements are being madeFluorescence filter/channels to No example given accommodate DAPI (UV)/Cy3/Alexa555, and Cy5/Alexa 647 Objectives based on camera resolutionOlympus UPLSAPO series described below objectives Monitoring/CalibrationStandard slide to provide reference for Omega optical fluorescencemicroscope standardization. [Optional] reference slide (blue; XF900);Ability to measure incoming light [Optional] Exfo NIST traceableintensity to microscope (in Watts) radiometer (part no. P010- 00200)Camera CCD monochromatic capability, 8 or Optronics QuantiFire XI CCD 12bit resolution camera (2048 × 2048 pixels, 7.4 μM/pixel) coupled withOlympus UPLSAPO 20X objective Field of view size Pixel size objectivemagnification Calculation for example (Camera/Objective combinationwhich provides field-of- hardware: (7.4 μM) * (2048)/ combination) viewsize between 671 μM and 888 μM. (20) = 758 μM field of view Field ofview size calculation (for cameras with rectangular CCDs, values must becalculated for both dimensions): (CCD pixel size) * (number of CCDpixels)/(objective magnification) Acquisition exposure Images must beacquired at optimal — exposure settings such that image pixels are notsaturated, yet intensity dynamic range is maximized Computer, monitor,Windows XP Professional (SPS) — keyboard, mouse equipped with a DVD-ROMdrive. 20″ monitor for image visualization.

It will be realized by one skilled in the art, that one or more of thesteps of obtaining the various correction factors: CC, LS, OP can beobtained automatically or at least semi-automatically with theassistance of a processor, such as computer. Alternatively or inaddition, one or more of the steps used in determining standardizedtarget image data and/or a quantitative measure therefrom can beautomated, for example, with the assistance of a processor. For example,such a processor can be implemented by a computer executingpre-programmed instructions. Such automation facilitates elimination ofthe inherent variability of pathologist-based scoring.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it should be apparent thatunique operational features have been described. Although particularembodiments have been disclosed herein in detail, this has been done byway of example for purposes of illustration only, and is not intended tobe limiting with respect to the scope of the appended claims whichfollow. In particular, it is contemplated by the inventors that varioussubstitutions, alterations, and modifications may be made to theinvention without departing from the spirit and scope of the inventionencompassed in the appended claims. For instance, the choice ofmaterials for the filter, the ordering of measurement and analysissteps, and the configuration of the filters, stage, and excitationsource employed is believed to be matter of routine for a person ofordinary skill in the art with knowledge of the embodiments describedherein.

1. A calibration instrument for sampling illumination of an excitationlight source of a microscopy system, comprising a calibration surfacepositioned along an optical path to substantially uniformly scatterillumination from the excitation light source toward a detection portionof the microscopy system.
 2. The calibration instrument of claim 1,further comprising: a dichromatic mirror positioned to reflectillumination from the excitation light source along an optical paththrough an objective toward a target sample and to transmit at least aportion of illumination from the target sample toward a detectionportion of the microscopy system, wherein the calibration surfacetemporarily blocks the optical path between the dichromatic mirror andthe objective during calibration and scattering a substantial portion ofthe reflected excitation light through the dichromatic mirror toward thedetector.
 3. The calibration instrument of claim 1, wherein thecalibration surface is opaque.
 4. The calibration instrument of claim 3,wherein the opaque calibration surface comprises filter paper.
 5. Thecalibration instrument of claim 3, wherein the opaque calibrationsurface comprises a substantially rigid surface.
 6. The calibrationinstrument of claim 5, wherein the substantially rigid surface comprisesa ceramic surface.
 7. The calibration instrument of claim 1, furthercomprising at last one neutral filter positioned to attenuate at leastone of the illumination from the excitation light source and theillumination from the target sample.
 8. The calibration instrument ofclaim 1, further comprising a housing configured to retain thedichromatic mirror, neutral filter, and calibration element in a fixedrelation with respect to each other.
 9. The calibration instrument ofclaim 8, wherein the housing is adapted for placement within aninterchangeable filter selection mechanism.